Stem cell therapy offers great promise in the search for an effective treatment for stroke, a leading cause of serious adult disability in the US afflicting nearly 800,000 Americans annually at a societal cost of billions of dollars. Preclinical data indicate that stem cell therapy, by targeting brain repair processes such as brain plasticity, has a therapeutic window of weeks to months implying it could benefit the majority of stroke patients. The mechanisms mediating stem cell-mediated recovery are, however, poorly understood. This will be important to dissect, as elucidating stem cell mechanisms of action will begin to delineate the molecular pathways of brain repair, and could also lead to improved efficacy and safety, and thus success, of stem cell therapy as it translates to the clinic. Our proposed study using cutting edge tools-TRAP, array tomography, and electrophysiology-will overcome existing technical barriers and enable us to address two significant gaps in our understanding of stem cell mechanisms: 1) What do transplanted stem cells express in vivo that promote brain plasticity and recovery? Based on previous work from us and others we hypothesize that human neural stem cells (hNPCs) elicit recovery by secreting paracrine factors that modulate brain plasticity. However, identifying these paracrine factors in vivo has been a challenge due to difficulties separating the hNPC and host expression profiles. We overcome this hurdle using the novel TRAP technique to separate transplanted hNPC mRNA from host brain mRNA. In Aim 1 we use TRAP plus microarray to generate the first in vivo transcriptional profile of hNPCs transplanted into the stroke brain and to identify candidate plasticity-modulating factors. We will then perform shRNA knockdown of these candidates in hNPCs and evaluate their effects on functional recovery and neurite plasticity using complementary in vitro and in vivo assays. 2) What synapse-level brain changes are elicited by stem cells that promote recovery? We reported that hNPCs enhance post-stroke plasticity by promoting axonal and dendritic sprouting. What is not known is how stroke and hNPCs affect synapse formation and function, which is ultimately where plasticity changes must occur to promote recovery. Such details were previously unattainable due to the complexity and minutia of synapses. The novel array tomography technique overcomes this hurdle enabling detailed structural analysis of individual synapses with cortical-layer specificity. Using this approach, we have identified a post- stroke increase in inhibitory GABAergic synapses and an hNPC-dependent increase in excitatory glutamatergic synapses. This has led to the hypothesis that hNPCs promote recovery by shifting the excitatory/inhibitory synaptic balance towards excitation. In Aim 2 we will use array tomography and electrophysiology, and our knockdown hNPC populations from Aim 1, to determine how hNPCs affect this balance, both in vitro and in vivo, and the resulting impact on post-stroke functional recovery.